【作者】 祝钧；

【导师】 吴海山；

【作者基本信息】 第二军医大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 背景内植物材料已在骨科得到广泛应用，内植物周围感染问题也日益突出。骨科内植物表面宿主免疫低下、磨损颗粒损害周围组织、细菌耐药、生物膜形成等导致这类感染极难治疗，也导致住院时间延长，增加患者痛苦，增加并发症和死亡风险，也造成医疗资源的巨大支出。许多研究致力于内植物表面改性，但往往存在抑菌能力欠佳、导致细菌耐药、妨碍骨整合等缺陷。如能研制出新型抗菌内植物表面，使其具备良好的抗菌能力与生物相容性，且可有效预防细菌粘附与生物膜形成，甚至可促进骨整合，将有一定的研究与应用前景。第一部分目的合成不同取代度的氯乙酰硫脲羧甲基壳聚糖衍生物（CCMC），研究其抑菌性能、细胞毒性以及对骨髓间充质干细胞（MSCs）增殖与分化的影响。方法1、以抑菌圈试验及抑菌率试验测试其对金黄色葡萄球菌、表皮葡萄球菌的体外抗菌性能；2、以MTT比色法研究其对MC3T3-E1细胞的细胞毒性；3、以MTT比色法测定其对MSCs增殖的影响；4、以碱性磷酸酶活性和骨钙素含量测定其对MSCs分化的影响。结果1、随着CCMC取代度的增高，其抑菌活性也逐渐增强；2、0.88取代度的CCMC具有体外细胞毒性，另两种取代度的CCMC没有体外细胞毒性；3、0.24与0.53取代度的CCMC对MSCs的增殖和分化均有一定促进作用，0.88取代度的CCMC对MSCs的增殖和分化均有一定抑制作用。第二部分目的测定不同取代度的CCMC的最小抑菌浓度及最小生物膜清除浓度，以及特定浓度下对金黄色葡萄球菌以及表皮葡萄球菌生物膜抑制率及对生物膜活性的影响。方法1、以细菌定量法测定不同取代度的CCMC的最小抑菌浓度，以比色法测定各取代度CCMC的最小生物膜清除浓度；2、以比色法测定各取代度CCMC在特定浓度下对三种生物膜的抑制率；3、以荧光染色比较各取代度CCMC对表皮葡萄球菌生物膜活性的影响。结果1、随着CCMC取代度的增高，最小抑菌浓度及最小生物膜清除浓度也逐渐减小；2、相同浓度下，取代度越高，CCMC对三种生物膜的抑制率越高，0.88与0.53取代度的CCMC在较小浓度下即可有效抑制生物膜生长；3、相同浓度下，取代度越高，CCMC对表皮葡萄球菌生物膜活性的抑制能力越强；随着浓度的增高，各取代度CCMC对表皮葡萄球菌生物膜活性的抑制能力越强。第三部分目的钛合金表面共价结合0.53取代度的CCMC（Ti-CCMC），通过与钛合金表面（Ti）的比较，研究这种钛表面抑制生物膜的能力、对成骨细胞粘附与增殖的影响，建立新西兰大白兔感染模型，研究Ti-CCMC对感染局部骨组织的影响与全身性影响。方法1、以荧光染色法比较Ti-CCMC与Ti在体外对生物膜活性的影响；2、以细菌定量法比较Ti-CCMC与Ti在体外对表皮葡萄球菌粘附的影响；3、比较Ti-CCMC与Ti对MC3T3-E1细胞粘附与增殖的影响；4、以细菌培养定量检测法测定钛棒周围骨组织内细菌含量；5、比较两组兔的体重变化，行血细菌培养、X线检查，研究两组兔全身性情况的差异；6、对两组兔股骨中段髓腔行革兰氏染色，比较两组细菌局部迁移的差异。结果1、Ti-CCMC体外对生物膜活性有明显抑制作用；2、Ti-CCMCC由一定的抑制表皮葡萄球菌粘附的能力；3、Ti-CCMC可促进MC3T3-E1细胞粘附与增殖；4、Ti组骨组织内见细菌生长，Ti-CCMC组未见细菌生长；5、两组兔体重比较无明显差异，Ti组术后X线检查发现股骨骨质破坏，Ti-CCMC组未见明显异常，未见骨溶解与透亮带；6、Ti组兔股骨中段髓腔行革兰氏染色，可见革兰氏蓝染的细菌散在，Ti组未见细菌。结论壳聚糖酰基硫脲衍生物具有良好的抗菌能力、生物相容性与骨整合能力，可作为骨科内植物表面抗菌的新型材料，具有一定的研究与应用前景。更多还原

【Abstract】 BackgroundWith the implant materials beening widely used in orthopedic surgery， infectionof the implant materials has become a difficult issue. Host immunosuppression overorthopedic implant surface, the surrounding tissue damaged by wear particles,bacterial resistance, biofilm formation, result in the extremely difficult treatment ofthis type of infection. It also leads to prolonged hospitalization, increased patientsuffering and the risk of complications and death, and also huge expenditure ofmedical resources. Many studies focus on the implant surface modification, bu oftenresult in poor antibacterial capacity, bacterial resistance and osseointegration defects.Novel antibacterial plant surfaces,which has good antibacterial ability andbiocompatibility, could preventing bacterial adhesion and biofilm formation, andeven promote bone integration, will be a certain amount of research and applicationprospects.Part ⅠObjectivesSynthesis of different degree of substitution of chloro acetyl thioureacarboxymethyl chitosan derivative (CCMC), to detect its antibacterial properties,cytotoxicity, and the impact of the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs).MethodsInhibition zone test and the inhibition rate test were used to determine theabilities of in vitro antibacterial properties of CCMC against Staphylococcus aureus,Staphylococcus epidermidis. MTT assay was used to determine the cytotoxic towardsMC3T3-E1cells. MSCs proliferation was also determined by the MTT assey.Alkaline phosphatase activity and osteocalcin content were measured for determiningMSCs differentiation.ResultsAlong with the increase of the degree of substitution of CCMC, the antibacterialactivity gradually increased. CCMC(0.88) had an in vitro cytotoxicity towards toMC3T3-E1cells, while the other two did not. CCMC(0.24) and CCMC(0.53) couldpromote the proliferation and differentiation of MSCs, while CCMC(0.88) had acertain inhibition on it.Part ⅡObjectivesTo determine the minimum inhibitory concentrationand the minimumbiofilm eradication concentrations of CCMC, as well as the impact of biofilmquantity and biofilm activity against Staphylococcus aureus and Staphylococcusepidermidis biofilm inhibition rate. MethodsBacterial quantitative method was used to determine the minimum inhibitoryconcentration of different degrees of substitution of CCMC. The minimum biofilmeradication concentration was determined also. The biofilm of each degree ofsubstitution CCMC inhibition rates were determined against three kinds of bacterials.The impact of CCMC on Staphylococcus epidermidis biofilm activity was determinedby the method of immunofluorescence assay.ResultsAs higher degree of substitution of CCMC, the minimum inhibitoryconcentration and the the minimum biofilm eradication concentration graduallydecreases. As in the same concentration, the inhibition rate of the CCMC againstthree biofilm increased along with the degree of substitution of CCMC. The degree ofsubstitution of0.88and0.53CCMC were effectively in a small concentration ininhibiting biofilm growth. Higher degree of substitution of CCMC had a strongerinhibition capacity on Staphylococcus epidermidis biofilm activity. With the increaseof the concentration of each substituted degrees CCMC, the inhibitory capacity of thebiofilm activity were stronger.Part ⅢObjectivesTitanium alloy surface covalently bonded with the0.53degree of substitution ofthe CCMC (Ti-CCMC). Through the comparison with the surface of titanium (Ti),the ability to inhibit the biofilm, the impact on the adhesion and proliferation ofosteoblasts were determined. A New Zealand white rabbit infection model was established. local impact of bone tissue and systemic impact of the animals weredetermined.MethodsImmunofluorescence assay was used to compare the ability of Ti-CCMC and Tiagainst biofilm activity.Bacteria quantitative method were used to compare the impactof Ti-CCMC and Ti on adhesion of Staphylococcus epidermidis and MC3T3-E1celladhesion and proliferation. Quantitative detection method was used for thedetermination of the bacterial content in the bone tissue around the titanium rod. Therabbits weight changes, Blood bacterial culture, X-ray examination, were used tocompare the differences in the two groups of rabbits. Differences between the twogroups of rabbits middle femur medullary cavity were compared between the twogroups of bacteria local migration by the method of gram stain.ResultsTi-CCMC had a significantly ability of inhibiting biofilm activity. Ti-CCMCcould also inhibit epidermal aureus adhesion.The Ti-CCMC could promote celladhesion and proliferation of MC3T3-E1.Rabbits in the Ti-CCMC group had nobacterial growth in the bone tissue surounding the rod. Rabbits weighing between thetwo groups showed no significant difference between the two groups. PostoperativeX-ray examination revealed the femur bone destruction in the Ti group, but theTi-CCMC group had no obvious abnormalities, osteolysis or Radiolucent line. Themiddle femur medullary cavity Gram stain in the Ti groups showed bacteria whichwere stained in blue.ConlusionsWith the good antibacterial ability, biocompatibility and bone integrationcapabilities. Chitosan acyl thiourea derivatives can be used as a new material of orthopedic implant surface. It deserves further research and has an applicationprospects.更多还原